A. Field of the Invention
This invention relates to a radiographic system and method for assessing the response of tissue in vivo to compounds, including therapeutic compounds.
B. Description of the Related Art
The ability to accurately assess the effects of compounds in vivo is essential in a wide range of pharmacological and related studies. Assessment of tissue response to compounds is particularly important in each stage of drug discovery, development, and clinical application. Noninvasive studies of tissue response may be particularly useful in the early validation of lead compounds during the drug discovery process, in clinical trials of potential therapeutic compounds, and in monitoring the efficacy of therapeutic compounds already used in clinical practice. These studies may include evaluation of therapeutic efficacy and detection of toxicity and other adverse side effects.
Evaluation of tissue response to compounds in vivo may initially be useful in the lead validation phase of the drug discovery process. Data that must be acquired during this early phase of drug discovery may be classified into three categories: pharmacodynamics, pharmacokinetics and toxicology. Pharmacodynamic studies include the evaluation of therapeutic efficacy in a targeted disease process, the relationship between compound dosage and therapeutic effect, and the duration of action of administered compounds. Pharmacokinetic studies include quantitative measurements of compound absorption, distribution, metabolism, and excretion (ADME). Toxicological studies include determination of gross systemic toxicity, damage to individual organs, and other adverse effects that may be caused by test compounds.
Pharmacodynamic, pharmacokinetic, and toxicological data are now frequently obtained by administering one or multiple doses of a test compound to an animal; killing the animal; and performing anatomical examination of body organs, histological examination of tissue, and analysis of body fluids. This approach to characterization of pharmacological activity has several significant limitations. First, anatomical and histological studies may not precisely reflect significant changes in the activity of relevant biochemical pathways in normal and abnormal tissue after administration of test compounds. Second, because animals must be killed for anatomical and histological studies, serial measurements cannot be performed on an individual animal during and after repeated administration of a test compound. Finally, processing of tissue for histological examination is labor-intensive, time consuming, and expensive, and often represents a serious rate-limiting step in applications such as high-throughput drug discovery.
For these reasons, a rapid, noninvasive method for assessment of tissue response to lead compounds during the early phases of the drug discovery process is highly desirable. The method may desirably enable quantitative measurements of changes in the activity of normal and abnormal biochemical pathways in tissue in response to administered compounds. The method may also desirably permit repeated, noninvasive measurements of the activity of these biochemical pathways in individual animals over long periods of time and over the course of multiple administrations of lead compounds. The method may also desirably allow data to be rapidly acquired and analyzed.
A second area of application for noninvasive assessment of tissue response in vivo is in clinical trials of new pharmaceutical compounds. A rapid, inexpensive method for assessment of tissue response might provide prompt accurate feedback on both efficacy and toxicity of test compounds in patients. Direct noninvasive assessment of tissue response might also provide data complementary to that obtained using measurements such as clinical chemistry studies of blood and urine.
A third application in which noninvasive assessment of tissue response to compounds should prove valuable is in the evaluation of efficacy and toxicity of therapeutic compounds already used in clinical practice. In many diseases processes, the response of abnormal or diseased tissue to therapeutic compounds may vary widely between individual patients. A noninvasive method that could provide prompt accurate feedback on the clinical efficacy of therapeutic compounds in individual patients should be of great value in selecting appropriate therapeutic agents, planning treatment regimens, and predicting outcome.
A number of approaches have been developed for noninvasive measurements of tissue response in vivo. These approaches have generally used techniques of nuclear medicine to generate images of a variety of tissue biochemical pathways. These imaging methods include positron emission tomography (PET) and single photon emission computed tomography (SPECT). A wide variety of radiopharmaceuticals have been successfully employed in PET and SPECT imaging studies. However, certain practical limitations of these modalities have reduced their economic feasibility and restricted their widespread use. These limitations include very high procedure costs, limited availability, need for dedicated imaging devices, and relatively low spatial resolution.
The spatial limitations of radiopharmaceutical-based imaging modalities are particularly problematic for lead validation studies in small animals. In these experimental models, the problem of low spatial resolution is more serious because of the small size of the subject relative to the fixed spatial resolution of the imaging modality. Alternative, relatively time-consuming and labor-intensive non-imaging-based methods have therefore been devised for metabolic measurements in small animals [Green L A et al.: J. Nucl. Med. 39: 729-734 (1998)].
The development and clinical use of anti-cancer chemotherapeutic agents is an exemplary pharmacological application that illustrates the potential utility of a method for inexpensive noninvasive assessment of tissue response in each stage of the process of drug discovery, clinical trials, and therapeutic use. In cancer chemotherapy, compounds are systemically administered to destroy or inhibit the growth of malignant cells in the body. Chemotherapeutic compounds are used to treat both primary malignancies and secondary metastases that may occur at distant sites in the body.
In the typical drug discovery process, initial lead validation in vivo of new potential chemotherapeutic compounds is now generally performed by means of manual anatomical measurements of implanted tumors in animals. Tumors or tumor cell suspensions are implanted or injected into the flanks of test animals. The tumors are often obtained from a foreign species, such as humans. After a variable period of growth, the size of the implanted tumor is determined. One or a combination of potential chemotherapeutic agents is then administered in a selected dosage protocol and serial measurements of changes in tumor size are performed over time. Tumor volume is estimated using two-dimensional measurements of tumor size which are manually obtained with calipers or a ruler. Shrinkage of the tumor is considered to reflect therapeutic efficacy of the test compound. [Geran R I et al.: Cancer Chemother. Res. 3: 51-61 (1972)]. Disadvantages of this method include the limited sensitivity and reproducibility of manual measurements, the difficulty of accurately determining tumor volume from two-dimensional surface measurements, the confounding effects of necrotic and scar tissue on tumor dimensions, and the inability to use tumor models that are typically localized deep within internal organs.
A rapid, noninvasive method for assessment of the response of malignant tissue to potential chemotherapeutic compounds should therefore provide significant advantages over methods currently used for evaluating therapeutic efficacy. Measurement of changes in biochemical pathways specifically associated with malignancy might provide a much more sensitive and rapid indicator of therapeutic efficacy than gross anatomical measurements of tumor size. A noninvasive method might also permit the use of tumor models in which the tumors are localized deep within internal organs. Such a method would also permit serial measurements of changes in tissue biochemistry without the need to kill an animal for histological examination. Measurements of tumor response in an individual animal over a long period of time and during administration of multiple doses of a test compound should enable assessment of secondary effects of the compound, including the likelihood of emergence of drug resistance. Both the initial and long-term therapeutic efficacy of a potential chemotherapeutic agent might thereby be evaluated in each individual animal.
Noninvasive assessment of tissue response to chemotherapeutic agents may also prove valuable in the clinical practice of oncological therapy. In a typical clinical setting, a course of chemotherapy may be administered for many weeks and often costs tens of thousands of dollars. The most common type of chemotherapy is known as adjuvant therapy, and is administered after the bulk of a malignant tumor has been removed by surgery. Starting at some time after the surgical procedure, one or a combination of chemotherapeutic agents is administered to the patient at regularly spaced intervals over a period of weeks or months.
Currently, the outcome of cancer chemotherapy varies widely in different tumor types and is unpredictable for any individual patient. Results of the therapy may vary from complete destruction of the tumor to its continued growth at an unchanged or accelerated rate. The response of any particular tumor to one or a combination of chemotherapeutic agents depends on many biological factors. These factors include the site of the tumor, the stage of tumor development at which therapy has been initiated, the genetic makeup of the tumor cells, and the degree to which the tumor is supplied by local blood vessels, or vascularized. Other, as yet unknown, variables probably also contribute to the widely differing responses of tumors to chemotherapy and the resulting outcome of treatment in individual patients.
In addition, the administration of chemotherapy is frequently accompanied by significant side effects to the patient including severe systemic toxicity. The physician must consider the probability of these side effects, and their impact on overall quality of life, when deciding on an appropriate chemotherapeutic regimen for each patient.
It is generally agreed among oncologists that, at present, cancer chemotherapy remains an art rather than an exact science. Selection of the optimal chemotherapeutic agents for treatment of a particular patient is now largely based on the general standards of treatment established in the medical literature, and on the past experience of the oncologist in treating similar tumor types.
Thus, it would be of great value in clinical practice to be able to rapidly and noninvasively assess the response of a malignant tumor in vivo to one or a combination of chemotherapeutic agents in each patient undergoing treatment. It would also be of great value to be able to predict the outcome of treatment based on quantitative measurements of tumor response after initiation of chemotherapy. Prompt feedback provided by early and frequent monitoring after the initiation of therapy would enable the oncologist to ascertain the effectiveness of the selected regimen and to modify it if the disease is not responding appropriately. Using noninvasive assessment of tissue response, the time necessary to determine the efficacy of a particular regimen may also be sharply reduced, lowering the risk that the cancer will have grown or metastasized in the interim.
A major area of application of radiopharmaceutical-based imaging for assessment of tissue response has been in the study of malignant tumors and their treatment. It is well known that almost all types of malignant tumors metabolize glucose at significantly higher rates than normal or benign tissue in the same organ [Hoh C K et al.: Semin. Nuc. Med. 27: 94-106 (1997); Conti P S et al.: Nuc. Med. Biol. 23: 717-735 (1996)]. Studies on changes in the glucose metabolic rate of malignant tissue in response to chemotherapy have been performed using the imaging modality of positron emission tomography (PET). In these studies, 18F-fluorodeoxyglucose (FDG), a positron-emitting radiopharmaceutical, was used to quantify rates of glucose metabolism. A number of these studies have demonstrated that the elevated rate of glucose metabolism in malignant tissue is reduced when the tissue is treated with chemotherapeutic agents [Wahl R L et al.: J. Clin. Oncol. 11: 2101-2111 (1993); Findlay M et al.: J. Clin. Oncol. 14: 700-708 (1996); Fischman A J: J. Clin. Oncol. 14: 691-696 (1996); Schulte M et al.: J. Nucl. Med. 40: 1637-1643 (1999)]. It has further been suggested in a number of studies that the relative reduction in glucose metabolic rate after treatment with a chemotherapeutic agent is reflective of therapeutic efficacy and may be predictive of the ultimate disease outcome. (Romer W et al.: Blood 91: 4464-4471 (1998); Bender H et al.: Hybridoma 18: 87-91 (1999)].
The validity of measuring changes in glucose metabolic rate to assess the efficacy of chemotherapy agents is supported by the finding that serial FDG measurements of untreated malignant tumors in humans conducted over a 10-day period before the initiation of chemotherapy remain within a narrow range and demonstrate high reproducibility between measurements [Weber W A et al.: J. Nucl. Med. 11:1771-1777 (1999)]. Subsequent changes in glucose metabolic rate as a result of administration of chemotherapeutic agents may thus be accurately quantified.
While PET imaging has demonstrated very high accuracy in the measurement of glucose metabolism in normal and malignant tissue, it suffers from disadvantages that diminish its utility for routine monitoring of chemotherapy. PET imaging devices are generally located only in large medical centers, and are therefore not available for widespread use. The procedure requires the synthesis of very short half-life radiopharmaceuticals. PET scans are very expensive, costing approximately $2500 per scan. In addition, the spatial resolution of PET is relatively low, and there is a consequent decrease in accuracy in imaging of tumors and metastases that are smaller than approximately 1 cm.
Similarly, SPECT imaging procedures are relatively expensive, and require the use of radiopharmaceuticals and a gamma camera. The spatial resolution of SPECT is also low, and the imaging technique is therefore inaccurate in imaging small tissue structures.
Thus, a need exists for an imaging modality capable of accurately evaluating the response of malignant tumors to chemotherapy in routine clinical application. The imaging modality may desirably be inexpensive and widely available, and provide the high spatial resolution necessary for imaging of small tumors and metastases. Such a method would be valuable in all phases of anti-cancer drug discovery, development, and clinical application.
Similarly, noninvasive assessment of tissue response to compounds in vivo may be useful in the discovery, development, and clinical application of a wide variety of other pharmaceutical compounds.
It is thus evident that there is a widespread need for improved assessment of tissue response to compounds, particularly therapeutic compounds, in live organisms. An ideal method may desirably be rapid, noninvasive, and inexpensive. The method may also allow the simultaneous evaluation of tissue anatomy and functional activity. A method with these advantages should improve the validation of lead compounds in vivo in the drug discovery process; provide valuable data in clinical trials on the efficacy of therapeutic compounds in patients; and provide rapid feedback regarding the response of individual patients to therapeutic compounds administered in clinical treatment. A method providing these advantages should satisfy important needs in all aspects of drug discovery, development, and clinical application.
The present invention is a radiographic system and method for assessing the response of tissue in a live organism to a compound. The invention provides radiographic images of the accumulation in tissue of a radio-opaque imaging agent that, in one embodiment, binds to a selected cellular target. Changes in the accumulation of the imaging agent in tissue after administration of a compound may be noninvasively monitored over time. Because the imaging procedure can repeatedly be performed on a live organism without detrimental effects, serial measurements may be performed to measure changes in tissue response to a compound over an extended period of time.
Because of these improvements and advantages, the present invention may be particularly useful for assessing the response of abnormal tissue, which is present in a disease process, to a potential therapeutic compound. The invention may thereby be used to evaluate the efficacy of lead compounds administered to animals in early stages of the drug discovery process. It may also be used to assess the response of tissue to compounds administered to patients during clinical trials. The invention may also be used to assess the efficacy of therapeutic compounds in individual patients undergoing clinical treatment. In this application, it may be used to rapidly and noninvasively provide feedback on the efficacy of a particular therapeutic regimen being administered to a patient. The physician may use this feedback on efficacy to modify the dosage or frequency of treatment, or entirely replace the therapeutic compound being used. The system and method may also be used to assess the response of tissue to compounds in a broad range of other in vivo studies, such as those involving the distribution and toxicology of compounds.
In one general form, the present invention is a method for assessing the response of tissue to a compound comprising:
(a) administering a compound to a live organism;
(b) administering a radio-opaque imaging agent that binds to a cellular target in the organism;
(c) generating an X-ray beam;
(d) illuminating the tissue with the X-ray beam; and
(e) acquiring a radiographic image of the tissue during illumination.
In another embodiment, a method includes:
(a) generating two or more X-ray beams with predetermined different energy spectra;
(b) illuminating the tissue with each of the X-ray beams;
(c) acquiring a radiographic image of the tissue during illumination by each of the beams; and
(d) performing a weighted combination of the acquired radiographic images to produce a single image.
The X-ray beams used in this embodiment may be quasi-monoenergetic or monoenergetic.
In one embodiment, the method further includes displaying variable proportions of radiographic density contributed by the imaging agent, by soft tissue, and by bone to the displayed image.
In one embodiment of the method, a selected interval is allowed for the administered compound to interact with tissue. The compound may optionally be repeatedly administered. In addition, serial radiographic images of tissue may be acquired at selected intervals.
In one embodiment, two or more of the acquired radiographic images may be compared. One method of comparison includes comparing the radiographic density of corresponding areas of displayed tissue on at least two acquired images.
In one embodiment of the invention, the radio-opaque imaging agent selectively binds, either covalently or non-covalently, to a cellular target within the organism. The cellular target may be a cellular structure, such as an organelle, an area of the cell, such as the cytoplasm, or a cellular molecule. The cellular molecule may be an enzyme, a non-enzyme protein, a coenzyme, a nucleic acid, or a lipid. In one embodiment of the invention, the cellular molecule is hexokinase. In one embodiment of the method, the cellular molecule is coenzyme A. In one embodiment of the invention, the radio-opaque imaging agent is bidirectionally cell membrane-permeable. In one embodiment, the imaging agent is cell membrane-permeable through passive diffusion.
In some embodiments of the invention, the imaging agent accumulates in abnormal tissue at a different rate or concentration than in normal tissue. In some embodiments, the imaging agent accumulates in malignant tissue at a different rate or concentration than in non-malignant tissue. In other embodiments, the imaging agent accumulates in abnormal myocardial tissue at a different rate or concentration than in normal myocardial tissue.
The administered compound may be chosen from potential or known therapeutic compounds, or other categories of compounds.
One embodiment of the invention is a method for radiographic assessment of tissue response to a compound (e.g. a method of measuring the therapeutic efficacy of a compound) wherein the method comprises:
(a) administering to a live organism a radio-opaque imaging agent that binds to a cellular target or is passively cell membrane-permeable;
(b) acquiring a first radiographic image of tissue in the organism;
(c) administering a compound to the organism after acquiring the first radiographic image;
(d) allowing an interval to elapse;
(e) acquiring a second radiographic image of tissue in the organism after administering the compound; and
(f) comparing the radiographic density of tissue displayed on the first and second radiographic images to assess the tissue response to the compound.
One embodiment further includes classifying the therapeutic efficacy of a compound based on the difference in radiographic density of tissue displayed on the first and second radiographic images.
In some embodiments of the invention, the imaging agent may accumulate in tissue in proportion to the glucose metabolic rate of the tissue. Changes in the glucose metabolic rate of tissue after administration of a compound, such as a therapeutic compound, may thereby be assessed. In some embodiments of the invention, the imaging agent accumulates in tissue in proportion to the rate of fatty acid metabolism of the tissue. Changes in the rate of fatty acid metabolism of tissue, particularly cardiac tissue, after administration of a compound, such as a therapeutic compound, may thereby be quantified.
The invention, according to one embodiment, is based on the well-established principle that the glucose metabolic rate of most types of malignant tumors is elevated compared with normal tissue in the same body organ. It has also been established that malignant tissue contains elevated levels of a number of the enzymes active in glucose metabolism.
Hexokinase is an enzyme which is particularly overexpressed in malignant cells. Hexokinase catalyzes the first step in glucose metabolism, which is the phosphorylation of glucose to glucose-6-phosphate. Quantitative studies have consistently demonstrated elevated levels of hexokinase in malignant tissue, with the increased enzyme level approximately proportional to the increased tissue glucose metabolic rate. Hexokinase is therefore an appropriate target enzyme for monitoring the glucose metabolic rate of malignant tissue.
Accordingly, one embodiment of the present invention utilizes a radiographic imaging agent that comprises a cell membrane-permeable, radio-opaque, high-affinity ligand for intracellular hexokinase. In this embodiment, a hexokinase substrate or inhibitor is linked to a non-radioactive, radio-opacifying moiety in a manner that facilitates efficient passage of the imaging agent across the outer cell membrane (plasma membrane), direct entry into the cytosol, and selective binding with high affinity to the substrate binding site of intracellular hexokinase molecules. The cell membrane-permeable property of the imaging agent molecules insures their direct entry into the cytosol, and the exit of unbound imaging agent molecules across the cell membrane. Thus, imaging agent molecules that have bound to hexokinase are retained within the cell for at least a few hours. Unbound imaging agent molecules exit the cell at a relatively rapid rate, decreasing background radio-opacity and increasing contrast in the radiographic image. Because of the much higher concentration of hexokinase in malignant cells relative to benign and normal cells, the imaging agent accumulates in malignant tissue at an elevated level relative to benign and normal tissue.
In an example of a system and method according to the invention, radiographic images using a radio-opaque imaging agent may be acquired before and after administration of a compound, and the images then compared.
A first radiographic procedure is initiated by administering the imaging agent to a live organism. After the imaging agent accumulates throughout body tissue during a time interval, the organism is appropriately positioned in relation to the X-ray source and image receptor, and radiographic images are acquired. Tissue that metabolizes glucose at a high rate, such as malignant tissue, will have accumulated higher intracellular levels of the imaging agent than tissue that metabolizes glucose at a lower rate, such as normal tissue. Because the imaging agent is radio-opaque, its differential accumulation causes corresponding differences in the absorption of the illuminating X-ray beam, which are manifested as differing levels of radiographic density on the radiograph.
A compound is then administered to the organism. A time interval may optionally be allowed to elapse to permit the compound to interact with tissue within the organism. A second radiographic imaging procedure is then performed. The radiographic density of corresponding areas of tissue displayed on the first and second images may then be compared.
In one embodiment of the invention, radiographic density contributed by the accumulated imaging agent is isolated from radiographic density contributed by soft tissue and bone. In this embodiment, the tissue being examined is sequentially transilluminated by X-ray beams with predetermined different mean energy spectra, and a separate radiographic image is acquired during transillumination by each beam. Using a predetermined weighting coefficient for each image, the image processing system performs a weighted combination of the acquired images to produce a single displayed image. The use of transilluminating X-ray beams with appropriate mean energy spectra together with appropriate weighting coefficients in the image processing procedure enables the cancellation on the displayed image of radiographic density contributed by soft tissue and bone. The remaining radiographic density present on the displayed image is contributed solely by differential intracellular accumulation of the radio-opaque imaging agent in malignant and normal tissue. Although this image of accumulated imaging agent is a functional image of tissue physiology, it is displayed with the high spatial resolution of a radiographic image, which may provide an anatomical image.
The system and method may also be used to generate radiographic images of tissue that combine both functional (biochemical) and anatomical (structural) information on a single image, with the two types of data in complete spatial registration. The viewer may interactively vary the proportion of radiographic density contributed to the displayed image by accumulated imaging agent, by soft tissue, and by bone. A functional image of tissue combined with a variable degree of a superimposed anatomical image in registration may thereby be displayed. The effects of a compound on both tissue biochemistry and anatomy may thereby be simultaneously assessed. The combination of functional and anatomical information on a single image, or, if desired, on a series of images, facilitates the precise localization of the accumulation of imaging agent in relation to nearby anatomical landmarks.
When used in the drug discovery process, the system and method may be used to quantify the response of tissue in an individual animal to one or more compounds administered over time. The variability between test animals in studies of efficacy and toxicity may thereby be significantly reduced. The invention may also reduce the need to perform labor-intensive and expensive gross anatomical studies of organs and histological examination of tissue.
An example of the utility of the present invention in the discovery, development, and clinical application of pharmacological compounds is in the field of cancer chemotherapy. A number of studies have suggested that measurement of changes in the glucose metabolic rate of malignant tissue after administration of chemotherapeutic agents can be used as reliable indicators of therapeutic efficacy [Bender H et al., Hybridoma 18: 87-91 (1999); Dehdashti F et al.: Eur. J. Nucl. Med. 26: 51-56 (1999); Price P, Jones T, Eur J Cancer 31A: 1924-1927 (1995)].
The present invention may be used to localize malignant tissue in vivo based on its elevated glucose metabolic rate, and to compare the glucose metabolic rate of malignant tissue before and after administration of an anti-cancer chemotherapeutic agent. The ability to obtain rapid and ongoing feedback on the response of malignant tissue to chemotherapeutic agents may be of significant clinical utility. Chemotherapy regimens that do not demonstrate therapeutic efficacy in a patient may be modified or halted, and a different regimen initiated. The time required to evaluate the efficacy of a particular therapeutic regimen may be significantly reduced, which may lower the risk of metastasis in the interim.
The present system and method may also be used to evaluate the efficacy of therapeutic compounds used in cardiac disease. The rates of glucose and fatty acid metabolism are often lowered in diseased cardiac tissue. The present invention may be used to localize diseased or damaged cardiac tissue in vivo based on its abnormally low glucose metabolic rate, and to compare the glucose metabolic rate of cardiac tissue before and after administration of cardiac therapeutic agents. Cardiac function may thereby be evaluated over time after initiation of a course of therapy, and the treatment regimen modified based on the results.
It will be apparent to one skilled in the art that the present invention may also be used to evaluate the response of tissue to a wide variety of compounds in studies unrelated to therapeutic efficacy. For example, the invention may be used in the assessment of toxicity of compounds. A decrease in the local tissue concentration of a particular cellular target in a body organ might be used as an indicator of compound toxicity.
The invention may be used for imaging tissue in particular body organs such as the breast, lungs, liver and colon, in single or multiple images, and for imaging large areas of the body or the whole body in single or multiple images. In addition, it will be apparent to one skilled in the art that with the appropriate modifications to the imaging method and imaging agents, the present invention may be used in computed tomography and other radiographic modalities.
The present invention is thus capable of noninvasively generating images for assessing the response of tissue to a compound, while providing the high degree of anatomical detail and spatial resolution characteristic of radiographic imaging.